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The origin of the antibacterial property of bamboo

a a a a
T. Afrin , T. Tsuzuki , R.K. Kanwar & X. Wang
a
Centre for Material and Fibre Innovation, Institute for Technology Research and
Innovation, Geelong Technology Precinct, Deakin University , Geelong , Australia
Published online: 01 Nov 2011.

To cite this article: T. Afrin , T. Tsuzuki , R.K. Kanwar & X. Wang (2012) The origin of the antibacterial property of bamboo,
The Journal of The Textile Institute, 103:8, 844-849, DOI: 10.1080/00405000.2011.614742

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 The Journal of The Textile Institute
Vol. 103, No. 8, August 2012, 844849

The origin of the antibacterial property of bamboo

T. Afrin*, T. Tsuzuki, R.K. Kanwar and X. Wang
Centre for Material and Fibre Innovation, Institute for Technology Research and Innovation, Geelong Technology Precinct,
Deakin University, Geelong, Australia
(Received 20 June 2011; nal version received 11 August 2011)

Bamboo is an eco-friendly and multifunctional plant. Bamboo clothing has recently entered the textile market with
a claim for its antimicrobial properties, but without scientic evidence. In this study, the antibacterial activity of
plant extracts from Australian-grown bamboo (Phyllostachys pubescens) is investigated. Bamboo extracts were
made using water, dimethyl sulphoxide (DMSO) and dioxane and their antibacterial properties were compared
against Gram-negative bacteria, Escherichia coli. It was found that the extract made in 20% DMSO aqueous solu-
Downloaded by [Deakin University Library] at 23:44 04 May 2014

tion showed weak antibacterial activity, whereas the extract made using 90% dioxane aqueous solution exhibited
strong antibacterial activity, even after 20 times dilution. The results indicate that antibacterial agents of P. pubes-
cens are located in lignin, not in hemicellulose or other water-soluble chemical components.
Keywords: bamboo; antibacterial property; lignin; E. coli

Introduction properties in bamboo has not been fully investigated.

Bamboo is a bast bre and considered as a green natu-
ral nanocomposites where cellulose nanobrils are
embedded in the matrix of lignin and hemicelluloses
(Afrin, Tsuzuki, & Wang, 2010; Rao & Rao, 2005).
Bamboo is well recognised for its multifunctionality and
eco-friendly nature and has been serving the daily needs
of over 1.5 billion of people for centuries (Austin, Levy,
& Ueda, 1970; Liese, 2009). The use of bamboo in
medicinal applications has a long history. It was shown
that the leaves of some bamboo species have an antioxi-
dative activity (Lu, Wu, Tie, Zhang, & Zhang, 2005).
Bamboos role in oral medicine is also portrayed where
the crude extracts of Polygonum cuspidatum roots
showed a wide range of antibacterial activities against
both Gram-positive and Gram-negative bacteria due to
the presence of phenolic compounds in its chemical con-
stituents (Shan, Cai, Brooks, & Corke, 2008). Some
researchers have also reported antibacterial activity of
bamboo charcoal (Yang et al., 2009) and bamboo vine-
gar (Sulaiman, Murphy, Hashim, & Gritsch, 2005).
Recently, bamboo clothing have entered the textile
industry and many commercial bamboo fabric prod-
ucts are claimed to be eco-friendly and antibacterial.
However, most of the claims are made by the industry
stakeholders where little scientic evidence was pre-
sented (Afrin, Tsuzuki, & Wang, 2009). In particular,
the compound(s) responsible for antibacterial
In some Asian countries, the antibacterial agent in
bamboo plants is identied as kun that represents a
hydroxyl functional group (OH) in a direct transla-
tion, but it fails to describe the actual chemical com-
pound and its location in bamboo.
Bamboo is a lignincarbohydrate compound that is a
glycoconjugate where hydrophobic lignin is chemically
bound to hydrophilic polysaccharides, such as cellulose
and hemicellulose (Koshijima & Watanabe, 2003). The
extraction of lignin is commonly carried out using aque-
ous dioxane solutions (Bjrkman, 1954). The extraction
of hemicelluloses is typically made in dimethyl sulphox-
ide (DMSO) (Al-Bakri & A, 2007). Hence, in the
present study, extraction of Australian-grown moso bam-
boo (Phyllostachys pubescens) was carried out in water,
DMSO and aqueous dioxane, and the antibacterial activ-
ity of the extracts was investigated to elucidate the loca-
tion of the chemical compounds(s) responsible for
antibacterial properties in bamboo.
Experimental
Microorganisms and media
Gram-negative bacterium, Escherichia coli (E. coli)-
ATCC 25922 was used as test organism. The bacterial
inoculums were prepared to obtain a bacterial
suspension in exponential growth of 8  108 colony

*Corresponding author. Email: taf@deakin.edu.au

ISSN 0040-5000 print/ISSN 1754-2340 online
Copyright 2012 The Textile Institute
http://dx.doi.org/10.1080/00405000.2011.614742
http://www.tandfonline.com
 The Journal of The Textile Institute
forming units ml 1 in 5 ml of nutrient broth (modied
Trypton soya broth from Oxoids). Trypton soya agar
(from Oxoids) was used as the nutrient agar for the
agar plates. The Atherton cyber series autoclave was
used for sterilisation and media preparation at 121C
for 20 min.
Materials and methods
Bamboo (P. pubescens) plant samples were purchased
from Earthcare Farm at Crystal Waters Permaculture
Village in Queensland, Australia. They were matured
culms and already dried. The bamboo was crushed
into ne powder to give the possibly highest surface
area while extracting with solvents. First, the raw
bamboo specimen was crushed into a powder form in
a vertical turret mill (Hafco, Super Power BM-52VF;
Downloaded by [Deakin University Library] at 23:44 04 May 2014
Hare and Forbes Machinery house, Melbourne, Aus-
tralia) and then shaker milling was further performed
in a 8000M mixer/mill (Spex, Metuchen, NJ, USA) in
a steel container with the steel balls of 0.91 cm in
diameter with the weight ratio of sample:ball = 1:60.
Time-dependent water extraction was done with
raw bamboo powder. Ten grams of bamboo is added
to 300 ml of deionised sterile water. The extraction is
carried out for 1, 3, 6, 12, 24 and 72 h. After extrac-
tion the solution was centrifuged (Eppendorf centri-
fuge, 5430R) and the supernatants were collected.
Ajax supplied the DMSO. It has been reported that
the DMSO itself has an antibacterial activity (Ansel,
Norred, & Roth, 1969). Therefore, the dependence of
DMSO concentration in water on the antibacterial activ-
ity was studied within the concentration range from 0 to
100%. It was found that 20% is the best concentration
to use for extraction, because the numbers of the
colonies were in between 30 and 300, suitable for
colony-counting. To make bamboo extracts, 10 g of
milled bamboo powder was immersed into 300 ml of
100% DMSO and was kept at room temperature for
72 h with continuous stirring, followed by ltering to
collect the supernatants, in which deionised sterile water
were added to make 20% DMSO aqueous solution.
Reagent grade dioxane was purchased from
Sigma-Aldrich (Sydney, Australia). The extraction was
carried out at room temperature by keeping 10 g bam-
boo in 300 ml aqueous dioxane solution (water:diox-
ane = 1:9 v/v) for 72 h with continuous stirring. The
powderliquid mixtures were then ltered and the
supernatant was collected. The dioxane was evapo-
rated to make bamboo extracts in water so that there
is no effect of the dioxane on the antibacterial activity.
This was considered as 100% solution of milled wood
lignin (MWL) and was further diluted with sterile
deionised water to obtain 50, 25, 10 and 5%
solutions.
845
The E. coli growth in nutrient broth was monitored
by the optical density measurements using an Asys
micro plate reader spectrophotometer at 550 nm (Expert
plus UV; Type: G020151, ASYS Hitech GmbH, Eugen-
dorf, Austria). Hundred microlitres of E. coli inoculum
was added into 5 ml of bamboo extracts (water, DMSO
extractions and MWL in water) and incubated for 18 h
at 37oC in a shaker oven. Twenty percent DMSO was
used as control for bamboo extracts in DMSO and ster-
ile water was used as control for water extracts and
MWL, respectively. After 18 h of incubation, 100 ll of
the E. coli and or extract mixtures were plated (three of
each) and incubated for further 18 h at 37oC. After
incubation, the plates were observed on the light box
and pictures were taken. A Ricoh 12 mega pixel camera
was used for the photography of the agar plates with
bacterial growth.
Physiochemical characterisation of bamboo powder
The morphologies of milled bamboo powder and
lignin extracts were studied by scanning electron
microscopy (SEM) using a Supra 55 VP. A Fourier
transform infrared spectroscopy (FT-IR) was carried
out to identify the chemical bonds with a Bruker Ver-
tex 70 spectrometer (Etllingen, Germany) and associ-
ated software OPUS 5.5. A Malvern Mastersizer 2000
particle size analyser (Worcestershire, UK) was used
to measure the particle size of the milled bamboo
powder by static laser light scattering, with water as a
dispersant. The amounts of cellulose, hemicellulose
and lignin are measured according to Chinese standard
method GB5889-8.
Results and discussion
Physical appearance of bamboo powders and MWL
The particle size of the bamboo powder after vertical
turret milling was around 500 lm in diameter.
Further milling in a shaker mill reduced the particle
size down to 520 lm as shown in Figure 1(a). The
Figure 1. SEM images of (a) milled bamboo powder and
(b) extracted bamboo lignin by using 90% aqueous dioxane.
 846 T. Afrin et al.
particle sizing by laser-light scattering indicated that
the average volume particle size was 30 lm. The
larger particle size observed by laser light scattering
may be caused by the fact that the particles swelled
during the measurements in water, while the SEM
image was taken for dried particles.
After extraction in 90% dioxane aqueous solution,
the dioxane was evaporated and the bamboo extracts
were collected in water that is termed as MWL. The
SEM image of MWL is shown in Figure 1(b). These
particles were found to be quite similar to the particles
isolated by Liese (1998). This observation gives the
indication of successful extraction of the gummy
material, i.e. lignin.
Chemical constituents
Downloaded by [Deakin University Library] at 23:44 04 May 2014
Figure 2 shows the chemical constituents of the Aus-
tralian-grown bamboo (P. pubescens) measured
according to Chinese standard method GB5889-8. Cel-
lulose, hemicellulose and lignin were identied as 53,
15 and 28%, respectively.
FT-IR spectroscopy was carried out on the
untreated bamboo samples to reveal the chemical
bonds, especially in the lignin region of the bamboo,
as shown in Figure 3. It has been indicated earlier that
the absorption peaks associated with lignin are located
in the range from 1500 to 1750 cm 1 (Yueping et al.,
2010). However, in this study, two major components
of lignin are guaiacyl and syringyl and their stretching
vibration rings are evident in a lower wavenumber
range at 1230 and 1160 cm 1, respectively. Also 1740
and 1675 cm 1 bands are identied as nonconjugated
carbonyl stretching and conjugated carbonyl stretch-
ing, respectively. The 1600, 1505 and 1425 cm 1
Figure 2. Chemical constituents of Australian-grown
bamboo (P. pubescens) measured according to GB5889-8
standard.
bands are aromatic skeletal vibrations (Buta, Zadrazil,
& Galletti, 1989; Sakakibara & Sano, 2001; Yueping
et al., 2010). The spectrum also shows the typical
cellulose nger print where 1050 cm 1 band is
assigned to complex vibrations associated with the
CO, CC stretching and COH bending in polysac-
charides (Rodrguez-Lucena, Lucena, & Hernndez-
Apaolaza, 2009; Yueping et al., 2010). The 1375 cm 1
band corresponds to the CH deformation in
cellulose and hemicellulose. CH deformation in cel-
lulose is evident in 898 cm 1 band (Pandey & Pitman,
2003).
Antimicrobial activity
Bamboo (time dependent) extracts in water
The antibacterial activity of bamboo extracts in water
is presented in Figure 4. It is evident that the bamboo
extracts in water could not inhibit or kill the growth
of E. coli as the plates of control and the bamboo
extracts in water have shown similar appearance in
the bacterial lawn. Therefore, it is clear that the anti-
bacterial compound(s) (if any) of bamboo (P. pubes-
cens) is not water soluble.
Bamboo extracts in 20% aqueous DMSO
Figure 5 shows photographical images of E. coli colo-
nies on Trypton soya agar that were plated with 20%
DMSO solution (control) and the bamboo extracts in
20% DMSO after incubations for 18 h. We found that
the colony size was signicantly larger on the control
(DMSO) plates than on the bamboo extracts plates. It
gives the indication of inhibition of bacterial growth.
However, the colony number was higher in the bam-
boo extracts plates than the control plates. DMSO has
earlier been reported to be bacteriostatic for E. coli at
20% concentration (David, 1972). It is possible that
the typical plant solvent DMSO could not properly
extract the antibacterial compound out from bamboo.
DMSO is also reported as the solvent for extracting
hemicellulose (Haimer et al., 2010), and therefore, it
is evident that the hemicellulose could not show
prominent antibacterial activity.
Bamboo extracts in water made using dioxane
The MWL (the bamboo extract in an aqueous dioxane
solution after removal of dioxane) was diluted with
water into v/v 100% (undiluted), 50, 25, 10 and 5%
and their antibacterial properties were tested against
E. coli. Figure 6 shows photographical image of bac-
terial plaques that were plated with control solution,
undiluted MWL and diluted MWL. The control plates
had a full lawn of bacteria, whereas no bacterial col-
 The Journal of The Textile Institute 847
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Figure 3. FT-IR spectra of untreated bamboo (P. pubescens).

Figure 4. Photographical image of bacterial plaques that were plated with the bamboo extracts in water: (a) control, (b) 1 h,
(c) 3 h, (d) 6 h, (e) 12 h, (f) 18 h, (g) 24 h, and (h) 72 h of incubation.

ony was evident on the plates with bamboo extracts.

Figure 5. Photographical image of bacterial plaques that
were plated with (a) 20% DMSO solution and (b) bamboo
extracts in 20% DMSO.
It was demonstrated that at the lowest concentration,
5% of bamboo extract in water was sufcient to
achieve 100% sterilisation rate against strong bacteria
such as E. coli. Since dioxane was evaporated after
extracting bamboo in aqueous dioxane (dioxane:
water = 9:1), there was no effect of dioxane on the
antibacterial activity. Moreover, dioxane is safely and
commonly used to prove the antibacterial activity of
the Schiff base metals (Johari, Kumar, Kumar, &
Singh, 2009). Therefore, it is evident that the bamboo
 848 T. Afrin et al.
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Figure 6. Photographical image of bacterial plaques that were plated with (a) sterilised deionised water and (b) 100%, (c)
50%, (d) 25%, (e) 10%, and (f) 5% MWL in sterile water.
(P. pubescens) lignin contains strong antibacterial
compounds.
The location of antibacterial agents in P. pubescens
From the above antibacterial test results, it is evident
that the antibacterial agents of P. pubescens are
located in lignin, not in hemicelluloses. The water-
insoluble nature of antibacterial compounds also sug-
gests that the antibacterial agents reside in lignin
which is almost insoluble in water (Walker, 2006).
The chemical constituent analysis proved the pres-
ence of a high amount of lignin (28%) in the bamboo
powder used in this study. In general, lignin is an aro-
matic gummy material composed of guaiacyl, syringyl
and p-hydroxyphenyl functional groups as well as p-
coumaric acid that is esteried in the polymer systems
(Higuchi, 1969). The lignin in softwood-like bamboo
is composed of coniferyl alcohol as the principal
monomer (Dimmel, 2010). Lignin is a complicated
network of polymers made of oxidative coupling of
three major C6C3 (phenylpropanoid) units with many
carbon-to-carbon and ether linkages and is formed by
dehydrogenative polymerisation of three lignin precur-
sors, p-hydroxycinnamyl, coniferyl and sinapyl alco-
hols (Xu, Sun, Sun, Fowler, & Baird, 2006; Zhanga,
Liua, & Suna, 2010). FT-IR spectroscopy in this study
revealed the presence of aromatic and carbonyl func-
tional chemical groups in bamboo. Some edible plant
extracts have shown antibacterial activity because of
the presence of phenolic groups (Alzoreky & Nakaha-
ra, 2003). Other studies reported the separation of
bioactive lignophenol antioxidants from bamboo lignin
and described their neuroprotective activity (Akao
et al., 2004; Ito et al., 2007). Zemek, Kosikova, Augu-
stin, and Joniak (1979) also depicted antibiotic effects
of synthetic compounds having guaiacyl and syringyl
structures that are related to the structure of native lig-
nin. As such, the existence of the aromatic and pheno-
lic functional groups in lignin may be responsible for
the antibacterial property of P. pubescens.
In order to produce antibacterial bamboo fabrics,
lignin components need to be retained into the bres
while processing raw bamboo into bre. However, cur-
rent methods to process bamboo plants into bres are
based on the regeneration principle where bamboo
plants are dissolved into solvents like alkali and carbon
disulphide to reconstruct cellulose-rich bres (Ryd-
holm, 1965), through which the functional chemical
compound like lignin is lost. Therefore, there is a
strong need for the development of new bre produc-
tion methods that enables the retention of lignin in the
nal bre products.
Conclusion
In this study, the origin of antibacterial property of
Australian-grown bamboo was investigated. The bam-
boo extract in the typical plant solvent DMSO to
extract hemicellulose showed the inhibition of bacterial
growth but could not kill the bacteria. The MWL
which was extracted in aqueous dioxane showed 100%
antibacterial activity even after extensive dilution.
Therefore, it is concluded that the antibacterial com-
pound of bamboo is located in lignin. FT-IR results
suggested that the antibacterial property may stem from
 The Journal of The Textile Institute
the aromatic and phenolic functional groups in lignin.
Further antibacterial studies against a Gram-positive
bacterium Staphylococcus aureus are under way.
Acknowledgements
Researchers would like to thank Dr Xin Liu and Dr Tiffany
Gunning at Deakin University for their help and support.
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